Age-related neurodegenerative diseases such as Alzheimer's disease (AD) and dementia are one of the largest societal challenges today. The World Health Organization estimates that costs for care of the elderly will continue to increase and that the number of diagnosed dementia cases will triple by 2050 (World Health Organization and Alzheimer's Disease International—Status Report (2012) DEMENTIA: A public health priority, WHO). The first treatments for AD were neurotransmitter modulators such as acetylcholine esterase inhibitors and NMDA modulators. These therapies became available at the turn of the millennium and still form the cornerstone for symptomatic relief of memory deficits related to dementia and AD. However, these drugs do not target the underlying causes of AD, accumulation of amyloid-β (Aβ) peptide and tau protein aggregates and associated loss of neuronal synapses and eventually neurons.
Longitudinal, community-wide studies of the elderly (Weiner, M. W. et al. (2014) ADNI; Breteler, M. M. et al. (1992) Neuroepidemiology 11 Suppl 1, 23-28; Launer, L. J. (1992) Neuroepidemiology 11 Suppl 1, 2-13) together with large genome-wide association studies (Lambert, J. C. et al. (2013) Nat. Genet. 45, 1452-1458) have shown that AD is a heterogeneous mix of dementias where up to 10 percent of the advanced AD patients lack amyloid pathology (Crary, J. F. et al. (2014) Acta Neuropathol. 128, 755-766). Furthermore, seminal pathological studies by Braak & Braak (Braak, H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12) demonstrated a clear correlation between the degree of neurofibrillary tangle pathology and cognitive state prior to autopsy. These observations have been reinforced by several investigators (Nelson, P. T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381), and in recent longitudinal biomarker studies, which indicate that cerebrospinal fluid (CSF) levels of tau and phospho-tau increase throughout early and late stages of the disease (Jack, C. R., Jr. et al. (2013) Lancet Neurol. 12, 207-216).
As indicated above, the microtubule-associated protein, tau, and its hyper-phosphorylated version, form the main constituent of intracellular neurofibrillary tangles, which are one of the main hallmarks of AD. Furthermore, specific genetic variants of tau are associated with familial forms of frontotemporal dementia (FTD). Appearance of tau pathology in AD occurs in a distinct spatial pattern, starting in the entorhinal cortex, followed by hippocampal and cortical areas (Braak, H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12). The specific stage of tau pathology also correlates well with cognitive abilities (Nelson, P. T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381; Braak, E. et al. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl 3, 14-22). Taken together, this evidence forms the basis of a tau-based hypothesis for AD. It entails that the intracellular accumulation of tau leads to microtubule degeneration and spinal collapse. As a result, communication between neurons malfunctions and cell death follows. Recently, it has also been shown that tau itself may form an endo-pathogenic species that can transmit neurodegeneration from one cell to the next (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11, 909-913).
Tau as an Endo-Pathogen
Clavaguera and colleagues have demonstrated that tau itself may act as an endo-pathogen (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11, 909-913). Low spin brain extracts were isolated from P301S tau transgenic mice (Allen, B. et al. (2002) J. Neurosci. 22, 9340-9351), diluted and injected into the hippocampus and cortical areas of young ALZ17 mice. The ALZ17 mouse is a tau transgenic mouse line which only develops late pathology (Probst, A. et al. (2000) Acta Neuropathol. 99, 469-481). The injected ALZ17 mice quickly developed solid filamentous pathology, and administration of immuno-depleted brain extracts from P301S mice or extracts from wild type mice did not induce tau pathology. Fractionation of the brain extracts in soluble (S1) and sarcosyl-insoluble tau (P3) (Sahara, N. et al. (2013) J. Alzheimer's. Dis. 33, 249-263) and injection of these into ALZ17 mice demonstrated that the P3 fraction is most competent in inducing pathology. It contains most of the intracellular hyper-phosphorylated filamentous tau. The majority of pathology could also be induced when injecting P301S extracts into the brains of wild type mice, but no NFTs were formed. In subsequent studies, Clavaguera et al. have shown that human tau extracted from post-mortem brain tissue of other tauopathies (Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD)) may also induce tau pathology in the ALZ17 model (Clavaguera, F. et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 9535-9540). Since the presentation of these data, several other tau seeding and spreading models have been reported (Ahmed, Z. et al. (2014) Acta Neuropathol. 127, 667-683; Walker, L. C. et al. (2013) JAMA Neurol. 70, 304-310). The main conclusion from these studies indicates a mechanism by which pathogenic tau in intracellular inclusions is secreted from the cell into the periplasmic space. The pathological tau material is then transported along the vesicular sheath in both anterograde and retrograde direction and subsequently taken up by neighboring cells by means of bulk endocytosis. This mechanism explains why the spread of pathology observed in human disease follows a distinct anatomical pattern. Intriguingly, peripheral administration of pathological tau may accelerate the formation of tau pathology in ALZ17 mice (Clavaguera, F. et al. (2014) Acta Neuropathol. 127, 299-301).
Relation Between Alpha-Synuclein and Tau Pathology
Alpha-synuclein is a member of a family of proteins including beta- and gamma-synuclein and synoretin. Alpha-synuclein is expressed in the normal state associated with synapses and is believed to play a role in regulating synaptic vesicle release and thereby affect neural plasticity, learning and memory.
Several studies have implicated alpha-synuclein with a central role in Parkinson's Disease (PD) pathogenesis. The protein can aggregate to form intracellular insoluble fibrils in pathological conditions. For example, synuclein accumulates in Lewy Bodies (LB) (Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synuclein gene as well as duplications and triplications of the gene co-segregate with rare familial forms of parkinsonism (Kruger et al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-7).
An important finding has been that alpha-synuclein can be secreted into and be present in plasma and cerebrospinal fluid (CSF). Several studies, for example by Pacheco et al. (2015) and others (Conway et al., (2000) Proc Natl Acad Sci USA, 97:571-576; Volles et al., J. Biochem. (2003) 42:7871-7878) have suggested that extracellular-synuclein plays a pathogenic role in the brain. They demonstrated that alpha-synuclein possesses neurotoxicity toward brain neuronal plasma membranes exposed directly to extracellular-synuclein oligomers. Another intriguing hypothesis based on the data of synuclein secretion is that a prion-like spread of alpha-synuclein underlies the progression of Parkinson's disease and other synucleinopathies (Lee et al., Hansen et al. (2011) J. Clin Invest 121:715-725). These finding have given rise to a hope that extracellular-synuclein could be targeted by immunotherapy (Vekrellis et al. (2011) Lancet Neurol 10:1015-1025) and be a potential treatment of alpha-synucleinopathies. In addition to mutations, alternative splicing of the alpha-synuclein gene and posttranslational modifications of the protein, such as phosphorylation, ubiquitination, nitration, and truncation can create alpha-synuclein protein forms that have enhanced capacity to form aggregated and/or toxic forms of alpha-synuclein (Beyer and Ariza, Mol Neurobiol. 2013 April; 47(2):509-24). However, the precise pathological species of alpha-synuclein in alpha-synucleinopathies remains unknown. Various misfolded/aggregated/secreted species ranging from oligomers to fibrils, and different post-translational modifications have been associated with toxicity but there is no consensus on which is most important, if indeed there even is a single toxic species.
The co-appearance of pathologies, for example Lewy bodies, Abeta plaques and neurofibrillary tangles in subsets of patients with PD or Lewy bodies in a subset of AD patients (Galpern and Lang, Ann. Neurol. (2008), 59: 449-458) has led to investigations of to what extent aggregation prone proteins can cross-seed each other. Alpha-synuclein and tau proteins have been reported to be able to induce fibrillization of each upon co-incubation in vitro (Giasson et al., (2003) Science 300: 636-640). In cellular systems there is both evidence supporting and not supporting a cross-seeding of tau with alpha-synuclein fibrils. Holmes et al., could not demonstrate a cross-seeding of tau with fibrils made from full-length alpha-synuclein in a FRET based tau reporter cell line (Holmes et al., (2014) PNAS doi/10.1073: E4376-E4385). Nor could Tau aggregation be induced with fibrillated full-length alpha-synuclein A53T or a PTA-precipitated (to enrich for fibrillated alpha-synuclein) brain sample from a multiple system atrophy (MSA) patient (Woerman et al., (2015) PNAS doi/10.1073: E4949-E4958. Others have reported that under some conditions alpha-synuclein can induce tau aggregation, for example alpha-synuclein fibrils made from N-terminal truncated (21-140) alpha-synuclein was shown to induce tau phosphorylation in QBI293 cells (Waxman and Giasson (2011) J. Neurosci 31: 7604-7618). In neuronal cultures full length fibrillated alpha-synuclein do seed tau aggregation. However fibrillated alpha-synuclein made from truncated alpha-synuclein (1-120 or 32-140) can through repeated self-seeding in cells (5 or 10% of fibrillated alpha-synuclein from each passage included as seeds in the fibrilization of the subsequent passage) (Guo et al., (2013) Cell 154: 109-117).
To our knowledge no studies has hypothesised that secreted oligomeric or fibrillated forms of alpha-synuclein could be a contributing factor in the early pathogenesis of AD or other tauophaties in general independent of visible alpha-synuclein inclusions. The reports that could demonstrate a cross-seeding of tau with alpha-synuclein have focussed on explaining why there in some PD patients, with manifest alpha-synuclein aggregation (Lewy bodies) are found neurofibrillary tangles. These studies speculate that the cross-seeding can take place in areas where there is a close physiological association of tau and alpha-synuclein deposits (Giasson et al., (2003) Science 300: 636-640; Waxman and Giasson (2011) J. Neurosci 31: 7604-7618; Guo et al., (2013) Cell 154: 109-117). The idea that soluble extracellular oligomeric/fibrillated forms of alpha-synuclein and not intracellular aggregates in the form of Lewy bodies or Lewy neurites are the important contributing factor in the generation of tau neurofibrillar tangles is new.
Alpha-Synuclein Immunotherapies
Antibodies binding to alpha-synuclein have been developed as potential therapeutic agents to treat synucleinopathies, also known as Lewy body diseases (LBDs). Synucleinopathies are characterized by deposition of intracellular protein aggregates microscopically visible as Lewy bodies (LBs) and/or Lewy neurites, where the protein alpha-synuclein is a major component (Jellinger, Mov Disord. 2012 January; 27(1):8-30; McKeith et al., Neurology (1996) 47:1113-24). Synucleinopathies include Parkinson's disease (including idiopathic Parkinson's disease) and Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease (PD), pure autonomic failure and multiple system atrophy (MSA; e.g., Olivoponto-cerebellar Atrophy, Striatonigral Degeneration and Shy-Drager Syndrome).
Several different antibodies to alpha-synuclein have been shown to have therapeutic effect in preclinical animal models. Both an antibody targeting an epitope involving alpha-synuclein residues 91-99 and antibodies targeting an epitope that involves alpha-synuclein residues 118-126 have been shown to have an effect on motor and cognitive deficits in transgenic mice (Games et al. 2014). The most advanced of these antibodies is a humanized antibody based on the mouse monoclonal antibody 9E4, which targets an epitope that involves alpha-synuclein residues 118-126, and which is now in clinical trials in phase I. Also an antibody that targets an amino-terminal epitope of alpha-synuclein has been shown to have possible therapeutic potential in preventing spreading and toxicity of pathology in a mouse prion like transfer model (Tran et al. 2014) and a C-terminal antibody 274 which targets an epitope that involves alpha-synuclein residues 120-140 (Bae et al. 2012) was also shown to have an effect in preclinical model on spreading of the pathology from cell to cell. In addition to these, antibodies targeting conformational species such as oligomers and fibrils of alpha-synuclein have been shown to be able to at least reduce the levels of these presumably toxic alpha-synuclein species (Lindtsröm et al. 2014 and Spencer et al. 2014). These conformational antibodies that lower alpha-synuclein oligomer levels in vivo, such as mab47 were also shown to target epitopes in the C-terminus of alpha-synuclein, from amino acid 121-125 (US20120308572). Other conformational, fibril and oligomer specific antibodies also target C-terminal sequences (Vaikath et al. Neurobiol Dis. 2015 Apr. 30; 79:81-99). Importantly none of these alpha-synuclein antibodies has been claimed to be able to prevent tau aggregation and as a consequence be able to potentially treat tauopathies.
In this invention we surprisingly discovered that aggregated/fibrillated alpha-synuclein can induce aggregation of Tau and that several antibodies generated to bind alpha-synuclein are able to prevent this aggregation. We show that a panel of different alpha-synuclein antibodies are all able to prevent aggregation of Tau in the cellular model: An antibody (GM37) that can bind to the presumed toxic alpha-synuclein fragment 1-119/122 (binding to amino acids 112-117 of alpha-synuclein) and neutralize this truncated form of alpha-synuclein, an antibody (2E6) that bind to amino acid 136-140 of alpha-synuclein, an antibody (GM63) that bind to amino acid 126-138 of alpha-synuclein and an antibody 9E4 that bind to amino acid 118-126 of alpha-synuclein. To support that fibrillary forms of alpha-synuclein may contribute to early AD pathology we demonstrate the presence of fibrillated alpha-synuclein in brains from AD patients independent of the presence of Lewy body pathology. This supports that soluble extracellular forms of fibrillated alpha-synuclein can potentially play a role in contributing to Tau-pathology in tauopathies such as all AD patients and not only in those characterised with visible alpha-synuclein aggregates (determined by brain imaging or post mortem staining).